Method for detection of protein comprising histidine-tag using immunochromatography

Abstract

The present invention relates to a method for the detection of a protein including a histidine-tag using immunochromatography. The detection method of the present invention uses immunochromatography using the polymer particle fixed with the histidine-tag, wherein a histidine-tag specific antibody alone is used to detect a histidine-tag conjugated protein (recombinant protein) quickly without using a target protein specific antibody. This method is also efficient in detecting and quantifying the histidine-tagged protein included in the sample without using an additional apparatus but quickly and accurately.

What is claimed is: 
     
       1. An immunochromatography kit for the detection or quantification of a histidine-tagged target protein, the kit comprising:
 (a) a nanoparticle, the nanoparticle having a histidine-tag and BSA fixed thereon, and 
 (b) an immunochromatography strip comprising a sample pad to receive a liquid sample, the liquid sample comprising the histidine-tagged target protein, 
 a measurement pad, and 
 an absorption pad to absorb the liquid sample via capillary action; 
 wherein the measurement pad consists of a single test spot on which an anti-his-tag antibody is spotted to detect or quantify a histidine-tagged target protein, wherein the histidine-tag on the nanoparticle binds to the anti-his-tag antibody on the test spot competitively with the histidine-tagged target protein; and a control spot on which an anti-BSA antibody is spotted to check reaction error, wherein the BSA on the nanoparticle, which does not bind to anti-his-tag antibody competitively with the histidine tagged target protein, binds to the anti-BSA antibody on the control spot, 
 and wherein binding of the nanoparticle to the anti-his-tag antibody or anti-BSA antibody produces color. 
 
     
     
       2. The immunochromatography kit according to  claim 1 , wherein the histidine-tag fixed on the nanoparticle comprises SEQ ID NO 1 or SEQ ID NO 2. 
     
     
       3. The immunochromatography kit according to  claim 1 , wherein a size of the nanoparticle is 50 to 400 nm. 
     
     
       4. The immunochromatography kit according to  claim 1  wherein the sample containing the histidine-tagged target protein is a cell extract. 
     
     
       5. The immunochromatography kit according to  claim 1 , wherein the nanoparticle is a polymer particle or a metal particle, wherein the metal particle is selected form the group consisting of gold, silver and white gold. 
     
     
       6. The immunochromatography kit according to  claim 5 , wherein the polymer particle is a cellulose nanobead. 
     
     
       7. A method for detection or quantification of a histidine-tagged target protein, said method comprising the steps of:
 (a) providing the immunochromatography kit according to  claim 1 ; 
 (b) mixing the nanoparticle having a histidine-tag and BSA fixed thereon with a sample containing the histidine-tagged target protein to form a mixture; 
 (c) and performing immunochromatography to detect or quantify the histidine-tagged target protein in the sample by adding the mixture to the sample pad of the immunochromatography strip and detecting the histidine-tagged target protein by observing color development resulting from conjugation reaction between the nanoparticle and the anti-his-tag antibody on the test spot of the measurement pad. 
 
     
     
       8. The method according to  claim 7 , wherein the histidine-tag fixed on the nanoparticle comprises SEQ ID NO 1 or SEQ ID NO 2. 
     
     
       9. The method according to  claim 7 , wherein the nanoparticle is a polymer particle or a metal particle, the metal particle selected from group consisting of gold, silver and white gold. 
     
     
       10. The method according to  claim 9 , wherein the polymer particle is a cellulose nanobead. 
     
     
       11. The method according to  claim 7 , wherein a size of the nanoparticle is 50 to 400 nm. 
     
     
       12. The method according to  claim 7 , wherein the histidine-tag fixed on the nanoparticle competes with the histidine-tagged target protein present in the sample. 
     
     
       13. The method according to  claim 7 , wherein the sample containing the histidine-tagged target protein is a cell extract. 
     
     
       14. The method according to  claim 7 , wherein at step (c), if the histidine-tagged target protein is present in the sample, conjugation reaction between the histidine-tag fixed on the nanoparticle and the anti-his-tag antibody on the test spot of the measurement pad decreases, resulting in a decreased color development, and if the histidine-tagged target protein is not present in the sample, the conjugation reaction between the histidine-tag fixed on the nanoparticle and the anti-his-tag antibody at the test spot of the measurement pad increases, resulting in increased color development. 
     
     
       15. The method according to  claim 7 , wherein at step (c), as concentration of the histidine-tagged target protein in the sample increases, conjugation reaction between the histidine-tag fixed on the nanoparticle and the anti-his-tag antibody at the test spot of the measurement pad decreases, resulting in decreased color development, and as the concentration of the histidine-tagged target protein in the sample decreases, conjugation reaction between the histidine-tag fixed on the nanoparticle and the anti-his-tag antibody at the test spot of the measurement pad increases, resulting in increased color development. 
     
     
       16. The method according to  claim 7 , wherein at step (c), the quantity of histidine-tagged target protein is determined by comparing the observed color development to a standard curve.